TET2 gene is mutated and/or deleted with high frequencies in multiple forms of myeloid malignancies including CMML, MDS, MPN and AML. The majority of the TET2 mutations lead to nonsense/frameshift, suggesting loss of function. Therefore, TET2 has been speculated to be a putative tumor suppressor gene that is strongly implicated in the pathogenesis of myeloid malignancies. Small hairpin RNA-mediated depletion of Tet2 in murine hematopoietic stem/progenitor cells (HSC/HPC) alters their cell differentiation and increase the proportion of HSC/HPC in culture, suggesting that Tet2 is important for regulating normal hematopoiesis. The objective of this project is to define the physiological function of Tet2 in vivo and in the pathogenesis of myeloid malignancies. We generated several Tet2-targeted murine models. Tet2- null mice displayed a phenotype resembling CMML at 3 months of age, and ~30% of these mice died by 10 months of age due to the progression to an erythroid/myeloid leukemia-like phenotype. Therefore, these Tet2-null mice allow us to model patients with myeloid malignancy. In a competitive reconstitution assay, Tet2-/- HSCs had an increased hematopoietic repopulating capacity. After transplantation of Tet2-/-, but not control BM cells, two of the seven recipients exhibited CMML phenotype similar to that of Tet2-/- mice. We, therefore, hypothesize that Tet2 acts as a tumor suppressor in myelopoiesis by regulating the behavior of HSC. Three specific aims are proposed: Aim 1: To prove Tet2 acts as a tumor suppressor gene in myelopoiesis by characterizing the phenotype of Tet2-/- and Tet2+/- mice. We will determine the hematological phenotype associated with the loss of Tet2 function in mice. The development of any type of myeloid malignancies at an elevated frequency in Tet2-null mice will add persuasive evidence to support Tet2 as a tumor suppressor gene in myelopoiesis. Aim 2: To define the cellular mechanisms by which loss of Tet2 function in mice leads to myeloid malignancies. We will examine if deletion of Tet2 induces a phenotype that is HSC autonomous by using serial transplantation of HSCs and mouse models with hematopoiesis-specific Tet2 inactivation. Aim 3: To define the molecular mechanisms by which Tet2 exerts its tumor suppressor function in myelopoiesis. We will identify potential genetic targets of Tet2 and Tet2-interacting proteins, which will allow us to unveil the regulatory network of Tet2 and pave a way for uncovering the mechanism by which Tet2 regulates hematopoiesis and exerts its tumor suppressor function. The completion of these studies will greatly improve our understanding of the role of Tet2 in normal hematopoiesis and pathogenesis of myeloid malignancies. This information could lead to the identification of novel molecular targets for the treatment of patients with myeloid malignancies. The Tet2 murine models also offer a biological context in which drugs and other therapies can be tested and developed.